Further studies on adenylate kinase by proton NMR of the protein and its substrate complexes to map the active site which has not yet been successful in the crystalline state. Work on this project has been deferred to await the arrival of the 360 MHz spectrometer (summer, 1977). 31P-NMR studies on enzyme-bound substrates of 3-PGA kinase will allow both substrates and both products to be observed. New studies will be aimed at mapping the active sites of the kinases under investigation in our laboratory and of the adenyl transferring enzyme, the aminoacyl tRNA synthetase, by using the paramagnetic Mn(II) probe with enzyme-bound substrates to determine distances of Mn(II) to each P. Both 180 and 360 MHz spectrometers as well as 100 MHz will be available for these experiments allowing frequency dependent measurements to be made. The 19F-NMR studies of the tRNA will include systematic attempts to assign the resonances such as by reversible modification as in the bisulfite reaction mentioned. Identification of the residues involved and biological activity determinations will be done by the methods of Schulman and Pelka (J. Biol. Chem. (1977), 252, 814-819). If the modified species is biologically active, its interaction with its cognate and non-cognate enzymes will be investigated since at least three 19F resonances in the bisulfite adduct are so well-resolved that resolution should be retained after binding to enzyme. Other methods will include paramagnetic probes and, if possible, hybrids of normal and 19F fragments of tRNA. A systematic comparison of the effect of metal ions and polyamines on the structure by 1H and 31P-NMR and enzymatic activity of f-tRNAMet with its cognate E. coli enzyme will also be undertaken. The hydrogen-bonded region of the NMR spectrum will also be investigated in the synthetase complex (trypsin digested form) which is now available in our lab. BIBLIOGRAPHIC REFERENCE: Interactions of Phospho- and Dephosphosuccinyl Coenzyme A Synthetase with Manganous Ion and Substrates. Studies of Manganese Complexes by NMR Relaxation Rates of Water Protons. Daniel H. Buttlaire, Mildred Cohn and William A. Bridger, J. Biol. Chem., 252, 1957-1964, (1977).